The use of enzymes to saccharify lignocellulosic biomass is typically performed after other physical and/or chemical methods of pretreatment and can be accomplished prior to or in conjunction with fermentation. Pretreatment breaks down biomass to allow access to the enzymes, which can then hydrolyse the remaining cellulose, hemicellulose, and pectin polymers. Most enzymatic saccharification are performed with commercially available cell-free extracts of fungal cultures, or in some cases, bacterial cultures, designed to provide predominantly cellulase, xylanase, or pectinase hydrolysis of the lignocellulose. The fungal enzymes typically have optima of 45° C. and pH 4.5, which can differ significantly from optimal fermentation conditions, especially when the ethanologen is a bacterium.
Cellulose degradation can occur via free, secreted enzymes or by enzyme complexes attached to the surface of microorganisms (a cellulosome). While anaerobic organisms typically possess cellulosomes, aerobic bacteria and fungi typically employ free enzymes. The degradation of cellulose is achieved through the action of three types of enzymes: endo-glucanases, cellobiohydrolases (or exo-glucanases), and β-glucosidases. Endo- and exo-glucanases cleave within or at the end of the glucan chain, respectively, and are classified based on both their structural fold and catalytic mechanism. Hydrolysis of cellulose by glucanases is catalyzed by two carboxyl groups in the active site and can either invert or retain configuration of the anomeric carbon. Enzymes that retain chirality use a double-displacement mechanism with a covalent enzyme-substrate intermediate while enzymes that invert chirality operate by a single-step concerted mechanism. β-glucosidases cleave cellobiose to monomeric glucose and are essential for overall cellulose degradation to glucose; accumulated cellobiose and/or glucose inhibit the activity of glucanases.
Hemicellulases are either glycoside hydrolases (GHs) or carbohydrate esterases (CEs), and are classified into families based on their activity and homology of primary sequence. GH enzymes are responsible for the hydrolysis of glycosidic bonds, while ester linked acetate and ferulic acids side chains are cleaved by CE enzymes. As the structure of hemicellulose is very heterogeneous, a wide array of enzymes is necessary for hydrolysis. Additionally, many hemicellulases have carbohydrate-binding modules in addition to catalytic domains; as much of the hemicellulose structure can be insoluble, the carbohydrate-binding modules play a role in targeting of the enzymes to the polymers.
Xylan is one major type of hemicellulose. Xylanases cleave the β-1,4 glycosidic bonds of the xylose backbone, while xylosidases hydrolyze resultant oligomers to monomeric xylose. Ferulic acid esterases and acetyl-xylan esterases cleave the ester bonds of ferulic acid and acetate side chains, respectively. Arabinofuranosidases hydrolyze arabinofuranosyl side chains from the xylose backbone and can have varying specificity as to the location of the arabinofuranosyl group. Finally, glucuronidases are responsible for the cleavage of glucuronic acid side chain α-1,2-glycosidic bonds.
A second form of hemicellulose is substituted β-mannan, such as galactomannan. Much like xylanases, β-mannanases are responsible for cleaving the mannose backbone to oligomers, which are then hydrolyzed to monomeric mannose by mannosidases. Side chain moieties, like galactose, are cleaved by respective GHs, and, in this case, by α-galactosidases.
Pectinases can be divided into three general activity groups: protopectinases, which act on insoluble pectic polymers; esterases, which de-esterify methyl and acetyl moieties from pectin; and depolymerases, which either cleave or hydrolyze glycosidic bonds with polygalacturonic acid polymers. Protopectinases are usually unnecessary for degradation of lignocellulose if physical and/or chemical pretreatment methods have been employed prior to enzymatic saccharification.
Pectin methylesterases are well described in bacteria and fungi and are responsible for the hydrolysis of the ester linkages from the polygalacturonic acid backbone. Pectin acetylesterases, which act in the same manner as pectin methylesterases to remove acetyl groups, have been described in plants and fungi; however, this type of enzyme has been found in only one bacterium, Erwinia chrysanthemi 3937. Pectin esterases are particularly important because many depolymerases cannot act upon methylated or acetylated pectin.
Pectin depolymerases act upon the polygalacturonate backbone and belong to one of two families: polygalacturonases or lyases. Polygalacturonases are responsible for the hydrolytic cleavage of the polygalacturonate chain, while lyases cleave by β-elimination giving a Δ4,5-unsaturated product. For pectin polymers with a rhamnogalacturonan-I backbone, other hydrolases are also necessary; rhamnosidases hydrolyze rhamnose from the backbone, and arabinofuranosidases and galactosidases cleave arabinose and galactose, respectively, from substituted rhamnose subunits.